A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and Si nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3-to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four-to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.

We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5′ and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.

We have analyzed the biochemical defect in a mutant line of mice that produces less than 1% of the normal level of serum transferrin. This mouse line (Hp) transcribes the transferrin gene in liver at the same rate observed in normal mice, but the steady state levels of transferrin mRNA sequences are less than 20% of normal. Further hybridization studies reveal that most of the transferrin mRNA sequences present in homozygous Hp mouse liver are in the form of a 5 kb nuclear precursor instead of the mature 2.5 kb transferrin mRNA seen in normal mice. Using several different exon and intron probes from the mouse transferrin gene, we have shown that the 5 kb RNA precursor retains the last two introns of the transferrin gene but that the 5′ and middle introns have been removed by processing. The defect in transferrin mRNA processing also extends to nonhepatic tissues and we find the same lack of mature mRNA and increased precursor accumulation in brain RNA. Since Southern blot analysis does not reveal gross changes in the structure of the transferrin gene in Hp mice, we suggest that the Hp defect is due to a small deletion or point mutation that either disrupts splicing signals or uncovers cryptic splice signals that interfere with processing of the last two introns in the transferrin gene. This Hp mouse line provides an opportunity to study the effects of transferrin deficiency on development and iron homeostasis.

Transferrin is an iron-binding protein that is expressed as a major product in liver and secreted into the plasma. To study the tissue-specific regulatory regions of this gene, the genomic mouse transferrin (mTf) gene was cloned and characterized by partial sequence analysis and S1 nuclease mapping of the transcriptional start site. Fusion genes containing the transferrin gene promoter and 5'-flanking sequences were ligated to the human growth hormone (hGH) gene and used to produce transgenic mice. A deletion construct containing the -581 to +50 region of the transferrin gene was sufficient to direct a high level of liver-specific expression resembling endogenous transferrin gene expression. Deletion to -139 base pairs of 5'-flanking sequence gave a construct which retained liver specificity, but the magnitude of expression decreased severalfold. These results demonstrate the presence of a liver-specific transcriptional element between -139 and +50 and suggest the presence of a distal element between -581 and -139 that can further increase expression. Surprisingly, fusion constructs containing -3 kilobase pairs (kb) of 5'-flanking sequence gave higher levels of mRNA in nonhepatic tissues than did either the -581 or -139 construct. Further studies indicated that the high levels of circulating hGH in these transgenic mice specifically induced the endogenous transferrin and albumin genes in liver and also stimulated the normally low levels of expression of the endogenous transferrin gene in brain, heart, kidney, and muscle. A mutated hGH gene that does not produce active growth hormone was fused to the -3- to +50-kb transferrin sequences to produce the -3-kb mTf-hGX construct. A liver-specific pattern of expression was observed in transgenic mice harboring the -3-kb mTf-hGX construct, and this mutated transgene was shown to be induced four- to sevenfold by either bovine or human growth hormone. These results demonstrate the presence of a growth hormone-responsive element between -3 and +50 kb in the 5'-flanking region of the mTf gene promoter.